Back to the Top
Hi group
We are working on assessing protein binding of a pyrethroid
insecticide in rat plasma.
We started with dialysis method, threre are two problems: 1. compound
is not stable in tris or phosphate buffer (for e.g by 6 h we lost
half the compound) and 2. did not see compound in plasma side,
because it sticks to the membrane (washed the membrane with a solvent
and analyzed). Cellulose membrane from spectrum labs with molecular
weight cut-off of 12,000 - 14,000 was used
We also used ultrafuge. First, we wanted to see whether the compound
passess through membrane. So, known amount of compound in buffer or
saline was taken through the method. WHile the compound was detected
(100%) in the buffer when added, but after centrifugation, nothing
was detected in the filterate. Cellulose membrane is purchased from
millipore corporation with molecular weight cut-off of 10,000.
Would increasing molecular weight cut-off help? Any suggestions and/
or references to overcome this hurdle?
thanks in advance
satheesh
S. SATHEESH ANAND, Ph.D
Postdoctoral associate
Department of Pharmaceutical and Biomedical Sciences
College of Pharmacy
University of Georgia
Athens, GA-30602
TEl#706-542-5401
FAX#706-542-5358
Back to the Top
The following message was posted to: PharmPK
Hi Satheesh,
If a compound is not stable [in buffer (as you observed) or plasma], or
shows NSB to the dialysis plate material or cellulose membrane, or has
solubility limitations, or does not reach equilibrium (e.g., slow
off-rate or covalent interaction) then equilibrium dialysis may simply
be an unsuitable method for obtaining reliable protein binding data. My
lab doesn't use a MWCO higher than 12-14K since some of the components
in plasma might pass through the pores (i.e., some components like AGP
have a lower mol wt than albumin).
Re. improving stability, have you assessed your compound's
characteristics (maybe acidified ACN extraction, esterase inhibitor,
etc., would help)?
If stability and NSB are issues, then an ultracentrifugation approach
might be suitable, since the assay time is shorter, there's no cellulose
membrane, reaching equilibrium isn't a factor, etc.
Regards,
Adrian Sheldon
In Vitro ADMET, Charles River Labs, Worcester, MA
Back to the Top
Hi SATHEESH,
Check out the following reference. It can help in your case.
Khurana M, Paliwal JK, Kamboj VP, Gupta RC. Binding of centchroman
with human serum as determined by charcoal adsorption method.
Int J Pharm. 1999 Dec 10;192(2):109-14.
PMID: 10567742 [PubMed - indexed for MEDLINE]
Vipul Kumar
Post Doc Associate
Pharmaceutics
University of Florida, Gainesville- FL-32610
USA
vipul kumar gupta
Pharmacokinetics and
Metabolism Div.
CDRI, LUCKNOW-226001
PharmPK Discussion List Archive Index page
Copyright 1995-2010 David W. A. Bourne (david@boomer.org)